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Objective:To evaluatetheeffectof transfectingangiotensinⅡreceptor(AT1)anti-sensenucleotide(AT1A)on theexpressionof subtypesof AngiotensinⅡ(AngⅡ)receptormRNA,andsynthesesof proteinand nucleicacidincardiomyocytes.Methods:AT1cDNAsequence(476bp)wasclonedwithRT-PCRandinsertedinto PcDNA3.1(5.4kb)anti-senselyto constructan intactplasmidcontainingAT 1 A(PAT 1 A).It was transfectedintothe culturedcardiomyocytes,whichwasidentifiedwithRT-PCRandWesternblot.Synthesesof proteinandnucleicacid weredeterminedwith 3 H-Leuand 3 H-TdRincorporation,mRNAexpressionsof AT 1 andAT 2 wereobservedwith RT-PCR.Transfectedandnontransfectedcardiomyocyteswerecomparedafterstimulatedfor24h by AngII1×10 -7 mol/L.Results:WeconstructedPAT 1 A successfully.AT 1 mRNAandproteinwereexpressedsignificantlylessin transfectedcardiomyocytesthanthatin thecontrol(P<0.01).AT 1 mRNAexpressionwas markedlydecreased,and AT 2 mRNAobviouslyincreased(P<0.01);butno apparentdifferencewas foundin 3 H-Leucine( 3 H-Leu)and 3 H-Thymidine( 3 H-TdR)incorporationbetweenthetransfectedandnontransfectedcardiomyocytesafterstimulated for24h of AngⅡ10 -7 mol/L(P>0.05).Conclusion:AfterblockedwithAT 1 A,expressionof AT 1 mRNAincultured cardiomyocyteswas markedlysuppressed,whileAT 2 mRNAwas up-regulatedat thesametime.Thisfactsuggests thatsynthesesof bothproteinand nucleicacidin cardiomycytesmediatedwithAng II couldnot be effectively interruptedsimplywithAT1Ablocking.
Objective: To evaluate the effect of transfecting angiotensin II receptor (AT1) on the expression of subtypes of Angiotensin II (Ang II) receptormRNA, and syntheses of protein and nucleic acid encoding myocytes. Methods: AT1 cDNA Sequences (476 bp) was cloned with RT-PCRandinsertedinto PcDNA3.1 (5.4 kb) anti-senselyto constructan intactplasmidcontainingAT1A (PAT 1 A). It was transfected with cultured cardiomyocytes, which was identified with RT-PCR and Westernblot. Syntheses of protein and nucleic acid weredetermined with 3 H-Leuand 3 H-TdRincorporation, mRNAexpressionsofA1andAT2wereobservedwith RT-PCR.Transfectedandnontransfectedcardiomyocyteswerecomparedafterstimulatedfor24hby AngII1 10-7 mol / L.Results: WeconstructedPAT 1 A successfully.AT 1 mRNAandproteinwereexpressedsignificantlyinfectedcardiomyocytesthanthatin thecontrol (P <0.01) .AT 1 mRNAexpressionwas markedlydecreased, andAT 2 mRNAobviouslyincreased (P <0.01); butno apparentdifference was found in 3 H-Thucidine (3 H-Leu) and 3 H-Thymidine (3 H-TdR) incorporationbetweenthetransfectedandnontransfectedcardiomyocytesafterstimulatedfor24h of Ang II 10 -7 mol / L (P> 0.05) .Conclusion: AfterblockedwithAT 1 A, expressionof AT 1 mRNAincultured cardiomyocyteswas markedlysuppressed , whileAT2 mRNAwasup-regulatedat thesametime.Thisfactsuggests thatsynthesesof bothproteinand nucleicacidin cardiomycytesmediatedwith Ang II couldnot be effectively interruptedsiat1Ablocking.